ABSTRACT Microbial-derived Short Chain Fatty Acids (SCFAs) have emerged as important mediators of ?Disease Tolerance,? a process that seeks to limit the collateral damage to host tissues that can accompany immune responses. Best understood in the gut, SCFAs produced by commensal bacteria act as signaling molecules to ?reprogram? immune cells to a more toleragenic state. However, this process can also be manipulated, as tumor cells use SCFAs to reprogram immune cells to limit inflammation and promote tumor growth. Whether pathogenic bacteria use SCFAs in a similar fashion to promote virulence is not well-understood. SCFAs are the principle end-products of the metabolism of the Gram-positive pathogen Streptococcus pyogenes. This bacterium can cause diseases at numerous mucosal and soft-tissue sites that range from asymptomatic (vagina), to self-limiting (pharynx) to necrotic (cutaneous). The immune response against the organism differs at each of these tissues and infection at certain tissues (e.g. pharynx) can result in autoimmune sequelae (rheumatic fever). The basis for this diversity of disease and immunity has not been clearly established. However, the ability of S. pyogenes to produce SCFAs suggests that reprogramming resulting in manipulation of disease tolerance may play a significant role. As a lactic acid bacterium, the fermentative metabolism of S. pyogenes reduces pyruvate via several alternative pathways: to lactate via lactate dehydrogenase and by two parallel arms of mixed acid fermentation, whose activities are modulated by oxygen, to produce additional SCFAs. Depending on growth conditions and substrates, S. pyogenes, will produce distinct mixtures of SCFAs. Since different types of immune cells vary in the specific SCFAs they respond to, this suggests that the differences in growth substrates and conditions encountered at diverse tissues will alter S. pyogenes SCFA composition to alter the types of immune cells modulated, which may contribute to tissue-specific differences in disease progression, severity and immunity. To test this hypothesis, we have constructed mutants, both singly and in every pair-wise combination, in the three pathways for pyruvate reduction. These constrain SCFA production to defined ensembles insensitive to changes in growth substrates. Preliminary analysis in a murine model of subcutaneous disease supports the central hypothesis, as some mutants are fully virulent, others show varying degrees of attenuation and most interestingly, some multiply to levels equivalent to wild-type, but produce markedly less tissue pathology. This latter behavior is a hallmark of the manipulation of a disease tolerance response. To more rigorously test the contribution of SCFAs to virulence, this proposal will expand analysis of these mutants to other tissue sites, will test their influence on immunity, their modulation of regulatory immune cell function and will catalog the principle growth substrates utilized and SCFA products produced by S. pyogenes at different tissues.